A small number of anti-CD3 molecules on dendritic cells stimulate DNA synthesis in mouse T lymphocytes

Resting T cells enter cell cycle when challenged with anti-CD3 mAb and accessory cells that bear required Fc receptor (FcR). Presentation of anti-CD3 is though to be a model for antigens presented by accessory cells to the TCR complex. We have obtained evidence that the number of anti-CD3 molecules that are associated with the accessory cell can be very small. We first noticed that thymic dendritic cells and cultured, but not freshly isolated, epidermal Langerhans cells (LC) were active accessory cells for responses to anti-CD3 mAb. DNA synthesis was abrogated by a mAb to the FcR but not by mAb to other molecules used in clonally specific antigen recognition, i.e., class I and II MHC products or CD4 and CD8. The requisite FcR could be identified on the LC but in small numbers. Freshly isolated LC had 20,000 FcR per cell, while the more active cultured LC had only 2,000 sites, using 125I-anti-FcR mAb in quantitative binding studies. Individual LC had similar levels of FcR, as evidenced with a sensitive FACS. FcR could not be detected on T cells or within the dendritic cell cytoplasm, at the start of or during the mitogenesis response. When the response was assessed at 30 h with single cell assays, at least 20 T cells became lymphoblasts per added LC, and at least 8 T cells were synthesizing DNA while in contact with the LC in discrete cell clusters. To the extent that anti-CD3 represents a polyclonal model for antigen presentation to specific T cell clones, these results suggest two conclusions. First, only 200-300 molecules of ligand on dendritic cells may be required to trigger a T cell. Second, the maturation of LC in culture entails \u27sensitizing\u27 functions other than ligand presentation (anti-CD3 on FcR) top clonotypic T cell receptors

Distinct features of dendritic cells and anti-Ig activated B cells as stimulators of the primary mixed leukocyte reaction

Highly enriched populations of B lymphoblasts have been isolated after culture with anti-Ig-Sepharose and compared with dendritic cells as stimulators of CD4+ T cells in the murine MLR. The two populations clearly differed in phenotype; anti-Ig blasts were FcR+, B220+, 33D1-, while dendritic cells were FcR-, B220-, 33D1+. However, as MLR stimulators, they shared many common features. Both cells (a) expressed comparable levels of class II MHC products; (b) independently stimulated the primary MLR and the production of several T derived lymphokines including IL-2 and IL-4; and (c) were comparable in stimulating freshly sensitized T cells. However, the relative potencies of dendritic cells and anti-Ig blasts as primary MLR stimulators varied in a strain-dependent fashion. Only anti-Ig blasts could stimulate across an Mls barrier, being at least 100 times more active in stimulating Mls-mismatched, MHC-matched T cells, relative to syngeneic T cells. In contrast, dendritic cells were 10-30 times more potent than anti-Ig blasts when stimulating across an MHC barrier and were likewise more effective in binding MHC-disparate T cells to form the clusters in which the MLR was generated. Dendritic cell-T cell clustering was resistant to anti-LFA-1 mAb, while B blast-T cell clustering was totally blocked. Thus, anti-Ig B lymphoblasts and dendritic cells, two cell types which differ markedly in phenotype, also differ in efficiency and mechanism for initiating responses in allogeneic T cells

A small number of anti-CD3 molecules on dendritic cells stimulates DNA synthesis in mouse T lymphocytes

Romani, N., Inaba, K., Puré, E., Crowley, M., Witmer-Pack, M., and Steinman, R.M. A small number of anti-CD3 molecules on dendritic cells stimulates DNA synthesis in mouse T lymphocytes. J. Exp. Med. 169: 1153-1168, 1989https://digitalcommons.rockefeller.edu/historical-scientific-reports/1022/thumbnail.jp

Antigen processing by epidermal langerhans cells correlates with the level of biosynthesis of major histocompatibility complex class II molecules and expression of invariant chain

Two prior studies with a small number of T cell lines have shown that the presentation of native protein antigens by epidermal Langerhans cells (LC) is regulated. When freshly isolated, LC are efficient antigen-presenting cells (APC), but after a period of culture LC are inefficient or even inactive. The deficit in culture seems to be a selective loss in antigen processing, since cultured LC are otherwise rich in major histocompatibility complex (MHC) class II products and are active APC for alloantigens and mitogens, which do not require processing. We have extended the analysis by studying presentation to bulk populations of primed lymph node and a TT hybrid. Only freshly isolated LC can be pulsed with the protein antigens myoglobin and conalbumin, but once pulsed, antigen is retained in an immunogenic form for at least 2 d. The acquisition of antigen, presumably as MHC-peptide complexes, is inhibited if the fresh LC are exposed to foreign protein in the presence ofchloroquine or cycloheximide. The latter, in contrast, improves the efficacy ofantigen pulsing in anti-Ig-stimulated B blasts. In additional studies of mechanism, we noted that both fresh and cultured LC endocytose similar amounts of an antigen, rhodamineovalbumin, into perinuclear granules. However, freshly isolated LC synthesize high levels of class II MHC molecules and express higher amounts of the class II-associated invariant chain. Fresh LC are at least 5-10 times more active than many other cell types in the level of biosynthesis ofMHC class II products. These findings provide a physiologic model in which newly synthesized MHC class II molecules appear to be the principal vehicle for effective antigen processing by APC of the dendritic cell lineage. Another APC, the B lymphoblast, does not appear to require newly synthesized MHC class II molecules for presentation

Antigen processing by epidermal Langerhans cells correlates with the level of biosynthesis of MHC class II molecules and expression of invariant chain

Puré, E., Inaba, K., Crowley, M.T., Tardelli, L., Witmer-Pack, M.D., Ruberti, G., Fathman, G., and Steinman, R.M. Antigen processing by epidermal Langerhans cells correlates with the level of biosynthesis of MHC class II molecules and expression of invariant chain. J. Exp. Med. 172: 1459-1469, 1990https://digitalcommons.rockefeller.edu/historical-scientific-reports/1028/thumbnail.jp

Hyaluronan and CD44 antagonize mitogen-dependent cyclin D1 expression in mesenchymal cells

High molecular weight (HMW) hyaluronan (HA) is widely distributed in the extracellular matrix, but its biological activities remain incompletely understood. We previously reported that HMW-HA binding to CD44 antagonizes mitogen-induced S-phase entry in vascular smooth muscle cells (SMCs; Cuff, C.A., D. Kothapalli, I. Azonobi, S. Chun, Y. Zhang, R. Belkin, C. Yeh, A. Secreto, R.K. Assoian, D.J. Rader, and E. Puré. 2001. J. Clin. Invest. 108:1031–1040); we now characterize the underlying molecular mechanism and document its relevance in vivo. HMW-HA inhibits the mitogen-dependent induction of cyclin D1 and down-regulation of p27kip1 in vascular SMCs. p27kip1 messenger RNA levels were unaffected by HMW-HA, but the expression of Skp2, the rate-limiting component of the SCF complex that degrades p27kip1, was reduced. Rescue experiments identified cyclin D1 as the primary target of HMW-HA. Similar results were observed in fibroblasts, and these antimitogenic effects were not detected in CD44-null cells. Analysis of arteries from wild-type and CD44-null mice showed that the effects of HMW-HA/CD44 on cyclin D1 and Skp2 gene expression are detected in vivo and are associated with altered SMC proliferation after vascular injury

Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity.

The majority of chimeric antigen receptor (CAR) T-cell research has focused on attacking cancer cells. Here, we show that targeting the tumor-promoting, nontransformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single-chain Fv FAP [monoclonal antibody (mAb) 73.3] with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFN-γ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAP(hi) stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8(+) T-cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T-cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective, suggesting that further clinical development of anti-human FAP-CAR is warranted

Expression of Fibroblast Activation Protein in lungs of dogs with idiopathic pulmonary fibrosis and dogs with lung cancer

peer reviewedCanine Idiopathic Pulmonary Fibrosis (CIPF) is a progressive fibrotic interstitial lung disease of unknown aetiology, affecting predominantly the West Highland White Terrier (WHWT) breed. Currently, there is no curative treatment option available. Fibroblast Activation Protein (FAP) is a cell surface protease usually absent from normal tissue but specifically expressed in areas of active tissue remodelling such as in fibroblast foci in human idiopathic pulmonary fibrosis. In humans, it is also upregulated in various types of cancers, either in cancer-associated fibroblasts (CAFs), in cancer cells or in both, depending on the tumour type. The aim of this study was to assess the expression and localization of FAP in the lungs of WHWTs affected with CIPF, in comparison with WHWTs with healthy lungs and dogs with lung cancer. Post-mortem formalin-fixed lung biopsies prepared from WHWTs with CIPF (n=17, age from 10 to 15y), control WHWTs exempt from lung disease (n=4, age from 11 to 15y) and dogs from various breeds with lung cancer (n=8, age from 8 to 14y) were retrospectively used. Included lung neoplasia were adenocarcinomas (n=6), histiocytic sarcoma (n=1) and metastasized mammary adenocarcinoma (n=1). Immunohistochemistry (IHC) was performed using a rabbit anti-human FAP monoclonal antibody (#ab207178). An IHC staining index (absent, low, moderate or high) was attributed according to the percentage of positive cells combined with the staining intensity. FAP was identified in the lungs of 16 out of 17 (94%) WHWTs with CIPF (IHC index high, moderate, or low in respectively 10, 4 and 2 dogs), 2 out of 4 (50%) WHWTs with healthy lungs (1 of each moderate and low), and 7 out of 8 (88%) dogs with lung cancer (high and moderate in respectively 6 and 1 dogs). FAP was expressed by fibroblasts in areas of active fibrosis in CIPF and by CAFs (all types of cancer) and cancer cells (adenocarcinomas only, n=5) in lung tumours. Results of this study showed that FAP is moderately to markedly expressed by fibroblasts in most dogs affected with either CIPF or lung cancer. Accordingly, FAP should be considered as an interesting potential therapeutic target for both diseases and should encourage further studies in the future. The expression of FAP in healthy lungs of WHWTs should be further investigated, particularly in comparison with FAP expression in dogs from other breeds, as it might serve as an indicator of early fibrosis

Cytokines regulate the affinity of soluble CD44 for hyaluronan

AbstractCD44, a receptor for the extracellular matrix glycosaminoglycan hyaluronan, has been implicated in many adhesion-dependent cellular processes including tumor growth and metastasis. Soluble CD44 has been identified in the serum of normal individuals. Furthermore, tumor progression is often associated with marked increases in plasma levels of soluble CD44. Release of soluble CD44 by proteolytic cleavage (shedding) of membrane-anchored CD44 is likely to alter cellular responses to the environment due to modification of the cell surface and the potential for soluble CD44 to influence CD44-mediated hyaluronan binding to cell surfaces. Cellular activation is typically required to induce hyaluronan binding to cell surface CD44 but the affinity of endogenous soluble CD44 for hyaluronan remains unknown. In this study, we demonstrate that oncostatin M and transforming growth factor β1 (TGF-β1) which stimulate hyaluronan binding to HTB58 lung epithelial-derived tumor cells, also induce the release of soluble CD44. Interestingly, soluble CD44 released by oncostatin M-treated cells retained the ligand-binding properties of the membrane-anchored receptor. In contrast, soluble CD44 released from TGF-β1-treated HTB58 cells differed in its hyaluronan-binding capacity from cell surface CD44 expressed on TGF-β1-stimulated cells. These data indicate that the mechanisms that regulate the generation of soluble CD44 may also govern the binding of the released receptor to hyaluronan and therefore determine the impact on CD44-dependent physiologic and pathologic processes